To investigate the growth effect of branched oligosaccharides on the principal intestinal microorganisms, Bifdobacterium, Lactobacillus, Clostridium, Escherichia, Eubactemum, Enterucoccus, Staphylococcus and Bacteroides were cultivated on a medium containing branched oligosaccharides and panose. B. adolescentis, B. logum and L. aciduphilus grew effectively on the medium containing panose, while C. pe@igens, C. paraputrificum, Bac. fragilis and S. aurezds did not. The content of panose decreased greatiy in the culture broth of branched oligosaccharides of B, adolescentis, but it remained in the culture of C. perfringens. The results indicated that panose was consumed effectively by B. adolescentis, but not utilized by C. perfringens. 3. adolescentis still grew on the panose remained in the broth of mixed cultivation of B. adolescentis and C. perfn'ngens. Therefore, panose and branched oligosaccharides seem to promote selectively the growth of B. adolescentis in the human intestine.

In order to examine the viability depending on rehydration process and membrane injury of Nocardia mediterranei upon lyophilization, We labeled -thymidine in deoxyribonucleic acid of N. mediterrranei to obtain information on the mechanisms of injury caused by lyophilization. Suspensions of rehydrated cells were incubated with added DNase in a buffer solution. Extracellular radioactivity levels appeared to be high in the rehydrated solutions after lyophilization than freezing-thawing. Thus, the membrane systems were injured by lyophilization, but not ovenvhelmed. These considerations were confirmed by electron microscopy. In effects of rehydration, the cell membrane was seriously damaged by strong atmospheric pressure as soon as the inner ampule was opened, but this was not the case without admitting air under vacuum. N. rnediterranei cells, with no additives, were lyophilized and reconstituted without admitting air, virtually about 84% of the cells were viable.

Potato juice was found out to have a strong inhibition activity on the growth of Clostridium perf;nngens during work of foodstuffs for the improvement of human intestinal microflora. The anti-bacterial activity of the precipitated protein obtained from the potato juice in 70% ammonium sulfate solution was stable at the range of pH 4 to 10, whereas it was lost by a heat treatment at for 10 min. The minimal inhibitory concentration of the precipitated protein on the growth of C1. Pefingens was about 0.2 mg/ml. The potato protein also suppressed the growth of C1. butyrincm and Eubacterium iimosum, while it showed a promoting effect for the growth of Bifdobacterium bifidum, Bif: animalis, Lactobacillus plantarum and Lact. acidophitus. The potato protein was further purified by CM-Sepharose ion exchange column chromatography, Sephadex G-150 gel filtration column chromatography and SDS-polyacrylarnide gel electrophoresis. The purified protein(kCp) was proved to be a glycoprotein by PAS staining and its molecular weight was about 38.7 kd.

For the selection of powerful antagonistic bacterium for biological control of soil borne Eminia carotovora subsp. carotovora causing rot of vegetable, excellent strains (S4, S14, 565) were selected from 1,196 strains of bacteria which were isolated from rhizosphere in vegetable root rot-suppresive soil. Strains were identified to be Pseudomonas species with Api 20NE kit. Antagonistic substance was produced in 523 synthetic broth medium at pH 7~8 and during 3 days culture. The substance was stable in the pH range of 6 to 9. When the basal medium was supplemented with mannitol and sorbitol as carbon source and calcium chloride as metal salt, the production of the inhibitory substance was increased. The inhibitory acitivity was increased by the addition of fertilizer in soil. The isolated strains were resistant to the agricultural chemical such as benomyl and fosethyl-Al-folpet, and the antibiotics such as penicillin and lincomycin. We had found that Pseudomonas sp. S14 strain had a single plasmid. After treated with acridin orange for curing, we confirmed the existence of antagonistic gene in the chromosomal DNA.

Genomic DNA of Bacillus stearothemzophilus, which expressed alkalophilic and thermophilic xylanases, was partially digested with HindIII, cloned into pBR322, and subsequently transferred into the Escherichra coli HB101 cells. Three among 5, 000 transformants screened formed clear zones around their colonies. From the functional clones, three recombinant plasmids (pMG11, pMG12 and pMG13) had been isolated, and they were identified to carry the same 4 kb HindIII fragment originated from B. stearothemzophilus which was responsible for the xylanase activity. pMGl3, however, had the foreign DNA of opposite orientation compared to the other two recombinant plasmids. This recombinant plasmid gave much lower xylanase activity. B. stearothermophilus was observed to produce at least three xylanase activities as evidenced by the PAGE-xylan zymogram. The xylanase from E. coli HB101/pMG12 was judged to correspond to the largest among the three B. stearothermophilus xylanases observed in the zymogrom. The enzyme hydrolyzed xylooligosaccharides larger than xylotriose and degraded xylan to produce xylobiose and xylotriose as major products. The xylanase was considered to have trans-xylosidase activity, too.

Twenty-four bacterial strains among alkalophillic bacteria isolated from soil samples were examined for the presence of type II restriction endonuclease in aerobic culture. One strain was found to contain specific enzyme to cleave lambda DNA. The characteristics of this microorganism is the ability to grow well in alkalophilic and high temperature condition, that is at pH 10.3 and . This strain was tentatively identified to Bacillus alkaloPhilus subsp. halodurans when morphological, physiological and biochemical characteristics were examined. The enzyme was purified from crude extract by streptomycin sulfate, ammonium sulfate precipitation, which was followed by DEAE-cellulose and phosphocellulose ion exchange column chromatography, and the subunit molecular weight was about, 32,000 daltons by polyacrylamide gel electrophoresis containing 0.1% SDS.

An alkaline protease producing microorganism was isolated from soil and identified as Streptomyces griseus HC-1141. The optimum pH and temperature for the purified enzyme activity were 8.0 and , respectively. The enzyme was relatively stable in the pH range of 7.0-9.0 and at the temperature below . The activity of purified enzyme was inhibited by , , , and , whereas activated by and . -Amino caproic acid, 2,4-dinitrophenol and iodine did not show inhibitory effect on the activity of alkaline protease, but p-chloromercuribenzoic acid, ethylendiaminetetraacetic acid showed inhibitory effect on the enzyme activity. These result suggested that the protease was metalloenzyme, and require a reactive SH group for the activity. The reaction of this enzyme follows typical Michaelis-Menten kinetics with the value of M and the of g/min for casein. The activation energy for the alkaline protease calculated by Arrhenius equation was 3.643 kcal/mol. This enzyme hydrolyzed casein more rapidly than the hemoglobin and egg albumin.

This work was performed to investigate the possibility of using J96 hemolysin(Hly, Hly A) vaccine against urinary tract infecting Escherichia coli. Based on the known sequence of J96 hemolysin which was originally isolated from a pyelonephritis patient, ten 20-mer oligonucleotide probes were synthesized. Radioactive labelled 8 probes showed positive colony blots against most of the hemolysin producing wild type E. coli, while HA484 and HA661 showed 28.3, 71.7% positive blots, respectively. This result means that hemolysin genes are highly conserved. Also, 12 anti-Hly MABs(monoclonal antibodies) showed more than 90% positive immunoblots against secreted hemolysin from wild type E. coli. Especially, the result that MAB132 neutralized hemolysin from all of the wild type E. coli augments the idea that hemolysin will be effective as a vaccine.

An anticancer substance was prepared by ethanol precipitation of the hot water extract of culture mycelia of Conolus versicolor KFCC 30388. After 6 days of fermentation, the mycelia growth reached the peak and reducing sugar consumed almost all. HTCFA method has been employed for three human cancer cell lines, Hep-2(larynx cell), A-427 and Calu-3 (lung cell). Anticancer activities in A-427 and Calu-3 were 8.4 and 9.8% survival rate, respectively. The chemical analysis of the extract from the mycelia showed 42.2% of polysaccharide and 10.5% of protein. The polysaccharide consisted of five kinds of monosaccharides, L-glucose, D-glucose, galactose, mannose and xylose

Studies on the changes in rheological properties, molecular weight distribution and dextran yield after being reacted in lO%(w/w) sucrose concentration were performed with crude dextransucrase produced from Leuconostoc mesenteroides isolated from Sikhae. The reaction rate of dextran formation was monitored by sugar analysis with HPLC and by the changes in apparent viscosity. According to the periodate oxidation test, the dextran produced in this experiment was estimated to have 89% -(1->6) main linkages and 11% -(1->) side linkages. The rheological properties of the dextran solution formed changed with reaction time, and it was related to the changes in molecular weight distribution of dextran as determined by GPC analysis. As the reaction proceeded, the rheological behavior changed from Newtonian to non-Newtonian, showing Binghampseudoplastic and thixothropic flow behavior. The apparent viscosity of dextran formed solution increased with increasing reaction time, reached a maximum value of 2680 cP (=, ) by enzyme reaction for 8 hours, and then decreased. The temperature dependency of dextran formed solutions was well expressed by the Arrhenius equation and the activation energy reached a maximum value of 1.69 kcal/mole by enzyme reaction for 8 hours.

In order to develop more economic processes, continuous ethanol fermentation from starchy raw materials in a pilot scale multi-stage CSTR was investigated. Ethanol fermentation could be successfully operated for 30 days with naked barley and 60 days with cassava, respectively. Starchy raw materials used for this study were ground and passed through a 20-mesh sieve for low temperature cooking. Under the optimized conditions, the overall productivity of cassava was with an ethanol concentration of 9.51% (v/v), which was higher about 2 times than that obtained from a conventional batch system in industrial scale.

For the photoproduction of molecular hydrogen by photosynthetic bacteria in outdoor conditions, we constructed automatically controlled semi-continuous culture system. When the amount of hydrogen gas produced can be measured by a gas meter with a pulse generator, the same amount of substrate consumed for hydrogen production could be supplied by micro pump related with timers. Using the apparatus, we examined hydrogen production with immobilized cells of Rhodopseudomonas sPhaeroides B6 in outdoor conditions. In spite of severe fluctuation of weather and illumination, the culture was maintained under good control with regard to hydrogen productivity. It was possible to automate the semi-continuous outdoor culture of photosynthetic bacteria for hydrogen production.

A Streptomyces strain No. 182-27, which produced amino acid antimetabolite, was isolated from soil. During the course of screening for new amino acid antimetabolites from the culture broths of Actinomycetes, we found that the strain produced a substance active against Gram-positive bacteria and its activity was reversed by L-Ieucine on the synthetic minimal agar medium in the culture broth. The morphological and cultural characteristics serve to identify the producing organism strain 182-27 as the Streptomyces, although the species of this strain should be resolved in further studies. Fermentation was carried out in the synthetic medium at for 78 hours. The fermentation yield reached about 2 mg per liter of the broth. Purification was done by ion exchange resin, active carbon, silica gel column chromatography and obtained 20 mg of pure active substance from the 20 culture broth. The 182-27 substance was obtained as white powder, mp 18SoC. From the physicochemical characteristics of the substance, it was amino acid like substance but unknown about its chemical structure. It is active against some Gram-positive bacteria and reversed by L-Ieucine.

In order to improve the productivity of invertase by recombinant Saccharomyces cerevisiae containing SUC2 gene, the effect of amino acids and dissolved oxygen concentration on the gene expression was investigated. Optimal concentrations of leucine and histidine for cell growth and cloned gene expression were 0.03 gig and 0.04 gig, respectively, expressed as the ratio of amino acid/glucose. The lack or excess of leucine and histidine has inhibitory effect on cell growth and invertase expression. In batch culture, the less aeration was, the higher invertase activity was. In continuous culture at a dilution rate of 0.09 h 1 with controlled dissolved oxygen tension, invertase activity increased dramatically at DOT levels below 5% air saturation, and a maximum activity of 215.54 KUlg cell was obtained under unaerated condition.

In order to evaluate an enzyme-linked immunosorbent assay(ELISA) for practical use in detecting aflatoxin from cereals, we compared concentrations of samples contaminated artificially or naturally that were quantitated by the ELISA with those spiked or quantitated by HPLC. Cotton seed meals(19 items), rape seed meals(ll), soybean meals(9), and corns(3) imported from foreign countries were used as sample cereals. The standard curves of each cereal class showed that 1-100 ng/g of from cereals could be assayed by the ELISA. When artificially contaminated cereals were assayed by ELISA, the average recovery of AFB! from samples spiked to 3 ng/g and more was 138%(68-193%), although that spiked to 1 ng/g was somewhat high(268%). The average C.V. of recovery was 7.0%(0-22.2%). When naturally contaminated cereals were assayed, the concentrations of below 10 ng/g especially from rape seed meals quantitated by ELISA were much lower than those determined by HPLC. However, the concentrations of 10 ng/g and more from samples, except a few extraordinary samples. quantitated by ELISA were similar to those determined by HPLC, especially in case of cotton seed meals whose average recovery (ELISA/HPLC) was 153%. In conclusion, the ELISA was elucidated such as a practical tool to detect of 10 ng/g and more from cereals.